Patent Application
20230257693
The present disclosure relates to an evaluation technique for an enzyme activity.
In order to detect a specific component (substrate) in a liquid sample, a biosensor is employed. As a method for such a biosensor, the colorimetric method has been proposed. A biosensor using the calorimetric method (which will be referred to as a “calorimetric biosensor” or simply as a “calorimetric sensor” hereafter) includes a container or a flow path to hold a liquid sample (which will be simply referred to as the “container” hereafter) and a reactant member provided within the container. The reactant member includes an enzyme that reacts with the substrate contained in the liquid sample. The heat that occurs due to a reaction between the enzyme and the substrate is measured by means of a temperature sensor, so as to identify the kind or the concentration of the substrate.
It is necessary to fix the enzyme in the vicinity of the temperature sensor. Accordingly, with conventional techniques, the enzyme is mixed with a water-soluble light-sensitive resin containing polyvinyl chloride (PVC), and the substrate is coated with the mixed liquid of the enzyme and the resin. Subsequently, the mixed liquid is cured.
As a result of investigating conventional techniques for forming a reactant member by curing a mixed solution of an enzyme and a resin, the present inventor has come to recognize the following problem.
In a case of mixing an enzyme with a resin with a practical concentration for a calorimetric sensor, the mixed solution becomes cloudy. The cloudiness occurs due to agglomeration of the resin components. The reactant member formed by coating with such a cloudy mixed solution leads to non-uniformity of the resin density. Here, examples of non-uniformity include both non-uniformity of the density distribution in a given area and reproducibility (variation) in a case in which many samples are made using the same mixed solution. Uniformity of the density of the reactant member is important for the accuracy and reproducibility of measurement. Thus, improved uniformity of the reactant member is required.
With conventional techniques, an enzyme is immobilized by being captured within a mesh structure of the resin. Accordingly, if the resin has a non-uniform density distribution, the enzyme is strongly immobilized in a portion having high resin density. However, such an arrangement has the potential to involve a problem in that the enzyme dissolves into the liquid if the enzyme is captured in a region having a low resin density. Accordingly, this has the potential to involve a change in the enzyme density contained in the reactant member, leading to degradation in the measurement accuracy and reproducibility.
With the calorimetric sensor, in addition to the heat that occurs due to the reaction between the enzyme and the substrate, heat of wetting that occurs due to the liquid sample infiltrating into the resin is measured as noise. The heat of wetting occurs depending on the ease of infiltration of the liquid sample into the resin, i.e., depending on the resin density. Accordingly, if the resin has a non-uniform resin density for each sample of the reactant member, this leads to variation in the heat of wetting for each reactant member. This leads to degraded reproducibility.
It should be noted that such problems described above are by no means within the scope of common and general knowledge of those skilled in this art. Furthermore, it can be said that the problems described above have been uniquely recognized by the present inventor.
The present disclosure has been made in such a situation. Accordingly, it is an exemplary purpose of an embodiment of the present invention to provide a biosensor and a reactor with improved measurement accuracy and/or improved reproducibility.
An embodiment of the present disclosure relates to a biosensor reactor. The biosensor reactor includes: a container structured to hold a liquid sample containing a substrate; and a reactant member provided in an inner part of the container and containing an enzyme and a resin such that they are not mixed.
Another embodiment of the present disclosure relates to a manufacturing method for a biosensor reactor. The manufacturing method includes coating a holding sheet with a resin and curing the resin; and curing the holding sheet coated with an enzyme before the coating with the resin or after the curing of the resin.
It is to be noted that any arbitrary combination or rearrangement of the above-described structural components and so forth is effective as and encompassed by the present embodiments. Moreover, all of the features described in this summary are not necessarily required by embodiments so that the embodiment may also be a sub-combination of these described features. In addition, embodiments may have other features not described above.
Embodiments will now be described, by way of example only, with reference to the accompanying drawings which are meant to be exemplary, not limiting, and wherein like elements are numbered alike in several Figures, in which:
An outline of several example embodiments of the disclosure follows. This outline is provided for the convenience of the reader to provide a basic understanding of such embodiments and does not wholly define the breadth of the disclosure. This outline is not an extensive overview of all contemplated embodiments and is intended to neither identify key or critical elements of all embodiments nor to delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more embodiments in a simplified form as a prelude to the more detailed description that is presented later. For convenience, the term “one embodiment” may be used herein to refer to a single embodiment or multiple embodiments of the disclosure.
A biosensor reactor according to one embodiment includes: a container structured to hold a liquid sample containing a substrate; and a reactant member provided in an inner part of the container and containing an enzyme and a resin such that they are not mixed.
With this arrangement, the resin and enzyme are not mixed, thereby resolving a non-uniformity due to aggregation of the resin. With such an arrangement employing the biosensor reactor, this provides improved detection accuracy, thereby providing improved reproducibility.
For example, the “state in which an enzyme and a resin are not mixed” means a state in which the enzyme and the resin are separately immobilized. Examples of such a state include: a state in which the enzyme layer and the resin layer are arranged with an interface between them; and a state in which one of the enzymes or the resin is coated with the other.
In one embodiment, the enzyme and the resin may form a layered structure. With such an arrangement in which the first layer film is formed with one of the enzymes and the resin and the first layer is coated with the other, this is capable of preventing the resin and the enzyme from mixing. This provides a uniform resin density.
In one embodiment, the reactant member may include a holding sheet that is any one of fabric, paper, a porous member, and a mesh-structured member. The resin and the enzyme may exist in a state in which they infiltrate into the interior of the holding sheet. This allows the effective surface area of the reactant member to be increased. Such an increase of the surface area leads to an increase of an amount of heat and an increase of heat capacity, thereby facilitating detection by means of a temperature sensor.
In one embodiment, the reactant member may include a holding sheet. Also, the resin and enzyme may be layered on a surface of the holding sheet.
In one embodiment, the reactant member may have a hydrophilicity that is lower than that of a portion surrounding the reactant member. With this, such an arrangement is capable of generating a bubble at a portion that overlaps the reactant member. A bubble has a thermal conductivity that is lower than that of the liquid sample. Accordingly, this is capable of preventing the heat that occurs in the reactant member from escaping. This allows the change in temperature over time accompanying a reaction to be measured.
In one embodiment, the container may be provided with an opening. Also, the opening may be covered by a film. Also, the reactant member may be provided on an inner surface of the film. With such an arrangement in which the container and the film are configured as separable components, this allows the biosensor reactor to be manufactured in a simple manner. Furthermore, this allows the cost to be reduced.
In one embodiment, a surface layer of the film may be coated with a hydrophilic layer. With this, the hydrophilicity of the reactant member becomes relatively lower than that of the surrounding portions. This is capable of generating a bubble.
In one embodiment, the hydrophilicity of the container that faces the reactant member may be lower than that of the surrounding portions. With this, such an arrangement is capable of generating a bubble at a portion that overlaps the reactant member. A bubble has a thermal conductivity that is lower than that of the liquid sample. Accordingly, this is capable of preventing the heat that occurs in the reactant member from escaping. This allows the change in temperature over time accompanying a reaction to be measured.
A biosensor according to one embodiment may include: any one of the biosensor reactors described above; a temperature sensor structured to measure the temperature of the reactant member of the biosensor reactor; and a processing device structured to process an output of the temperature sensor.
A manufacturing method for a biosensor reactor according to one embodiment includes: coating a holding sheet with a resin and curing the resin; and curing the holding sheet coated with an enzyme before the coating with the resin or after the curing of the resin.
Also, the holding sheet may be any one of fabric, paper, a porous member, and a mesh-structured member.
Description will be made below regarding preferred embodiments with reference to the drawings. The same or similar components, members, and processes are denoted by the same reference numerals, and redundant description thereof will be omitted as appropriate. The embodiments have been described for exemplary purposes only, and are by no means intended to restrict the present invention. Also, it is not necessarily essential for the present invention that all the features or a combination thereof be provided as described in the embodiments.
In some cases, the sizes (thickness, length, width, and the like) of each component shown in the drawings are expanded or reduced as appropriate for ease of understanding. The size relation between multiple components in the drawings does not necessarily match the actual size relation between them. That is to say, even in a case in which a given member A has a thickness that is larger than that of another member B in the drawings, in some cases, in actuality, the member A has a thickness that is smaller than that of the member B.
The biosensor 100 includes a temperature sensor 110, a processing device 120, and a reactor 200.
The reactor 200 is configured as a test kit of the biosensor 100. The reactor 200 includes a container 210 and a reactant member 220. The container 210 houses a liquid sample (substrate solution) 4 including the substrate.
Specific examples of the liquid sample 4 include blood, urine, sweat, saliva, tears, etc., which are body fluids acquired from a human being. It should be noted that the liquid sample 4 is not restricted to the specific examples described above so long as the liquid sample 4 is a liquid of biological origin. For example, the liquid sample 4 may be a body liquid acquired from other animals, e.g., mammals such as dogs or cats, etc., birds, etc.
Also, the substrate is not restricted in particular. Specific examples of the substrate include glucose, uric acid, lactic acid, proteins, fat, creatinine, bilirubin, etc.
The reactant member 220 is provided in the inner part of the container 210. The reactant member 220 includes an enzyme 222 that can react with the substrate. Specific examples of the enzyme 222 include glucose oxidase, peroxidase, lactate oxidase, trypsin, lipase, creatininase, bilirubin oxidase, etc.
In addition to the enzyme 222, the reactant member 220 includes a resin 224 for holding and immobilizing the enzyme 222 in a stable manner. The material of the resin 224 is not restricted in particular. As the material of the resin 224, a water-soluble light-sensitive resin may be employed, examples of which include polyvinyl chloride (PVC), polyvinyl alcohol (PVOH), and BIOSURFINE (trademark)-AWP manufactured by Toyo Gosei Co., Ltd.
The reactant member 220 holds reaction heat that occurs due to the contact reaction between the enzyme 222 contained in the reactant member 220 and the substrate contained in the liquid sample 4. The temperature sensor 110 measures the temperature of the reactant member 220 of the reactor 200. The structure and the kind of the temperature sensor 110 are not restricted in particular. For example, a thermocouple may be employed.
A portion 212 of the container 210 to be provided with the reactant member 220 has a locally thin structure having a small thickness, which allows the temperature of the reactant member 220 to be measured from the exterior. The temperature sensor 110 is provided on the outer side of the container 210. The temperature sensor 110 measures the temperature of the reactant member 220 via this portion 212. This portion 212 is formed of a material and thickness that do not obstruct heat transfer. With this, there is no need to immerse the temperature sensor 110 in the liquid sample 4. This allows the temperature sensor 110 to be reused.
The processing device 120 processes the output of the temperature sensor 110 so as to estimate the presence or absence of the substrate or an amount of the substrate. As the calculation processing to be employed in the processing device 120, known techniques or techniques that will become available in the future may be employed. Accordingly, detailed description thereof will not be made in the present specification.
In the present embodiment, the reactant member 220 contains the enzyme 222 and the resin 224 in a state in which they do not mix with each other. From another viewpoint, the distribution density of the enzyme 222 in the reactant member 220 is substantially uniform. Furthermore, the density distribution of the resin 224 is also substantially uniform. It should be noted that
The above is the basic structure of the biosensor 100. In the biosensor 100, the reactant member 220 contains the resin 224 with a uniform density distribution as compared with conventional techniques in which a mixed solution of the enzyme 222 and the resin 224 is applied and cured. With such an arrangement in which the resin 224 is distributed with a uniform density distribution, such an arrangement is capable of suppressing variation in the heat of wetting for each sample, thereby providing improved reproducibility. Furthermore, this allows a region having a low density of the resin 224 to be reduced, thereby enabling prevention of dissolution of the enzyme 222. This is capable of suppressing change in the characteristics of the reactant member 220 over time.
Next, description will be made regarding a specific structure and manufacturing method of the reactant member 220.
The holding sheet 230A is not restricted in particular. Examples of the holding sheet 230A include non-woven fabric formed of Cupra fiber (Bemcot (trademark) PS-2) manufactured by Asahi Kasei corporation). For example, the holding sheet 230A has a circular structure having a diameter on the order of 1 mm and a thickness on the order of 30 μm. Also, a non-woven wiper such as Wypall manufactured by Nippon Paper Crecia Co., Ltd., may be employed as the holding sheet 230A. However, the present invention is not restricted to such an arrangement. Also, as the holding sheet 230A, non-woven fabrics other than those described above or fabrics other than non-woven fabrics may be employed.
As the holding sheet 230A, paper may be employed instead of such non-woven fabric. Specific examples of such paper that can be employed as the holding sheet 230A include non-woven paper, filter paper, blotting paper, Japanese paper, etc. Also, as the holding sheet 230A, a paper wiper such as Kimwipes or the like manufactured by Nippon Paper Crecia Co., Ltd. may be employed. However, the present invention is not restricted to such an arrangement. With such an arrangement in which the holding sheet 230A is formed of such fabric or paper, this allows the holding sheet 230A to be provided with a low cost.
Also, as the holding sheet 230A, a porous member or a mesh-structured member may be employed instead of such fabric or paper. Specific examples of the porous member that can be employed as the holding sheet 230A include a sponge having a continuous bubble structure. Also, examples of such a mesh-structured member that can be employed as the holding sheet 230A include a mesh member formed of woven thin metal wires having a size on the order of 10 μm.
Also, as the holding sheet 230A, absorbent sheet pieces using capillary action may be employed instead of fabric, paper, porous members, or mesh-structured members.
The resin and the enzyme are held such that they are distributed with a substantially uniform density in a state in which they infiltrate into the internal cavities 232 of the holding sheet 230A. It is difficult to specifically show the structure of the resin and the enzyme held inside the holding sheet 230A. Accordingly, the resin and the enzyme are not shown in
With such an arrangement employing the reactant member 220A shown in
Furthermore, the holding sheet 230A is formed of a material having internal cavities such as non-woven fabric, non-woven paper, a porous member, or the like. A large amount of the enzyme is stored inside the holding sheet 230A. As a result, this increases the contact area between the liquid sample 4 and the enzyme. This is capable of increasing the reaction heat ΔT due to the contact reaction between the substrate contained in the liquid sample 4 and the enzyme.
As shown in
As shown in
The film 240 preferably has a hydrophobic surface. This allows the solution dripped onto the holding sheet 230A to be prevented from spreading on the film 240. For example, as the film 240, an adhesive film may be employed. This is capable of immobilizing the holding sheet 230A using adhesion. Furthermore, such an adhesive surface is also hydrophobic, which is convenient.
Next, as shown in
Subsequently, the resin 224 is dried and cured by being irradiated with ultraviolet light.
In the reactant member 220A manufactured using this manufacturing method, the enzyme 222 is adhered to the surface of the fibers of the holding sheet 230A, whereby the enzyme 222 can be provided with a uniform density. Furthermore, the resin 224 uniformly infiltrates into the interior of the holding sheet 230A, whereby the resin 224 can also be provided with a uniform density. Furthermore, with such an arrangement in which, after the enzyme 222 is fixed, the resin 224 is cured, this allows the enzyme 222 and the resin 224 to be held in an unmixed state.
By making microscopic observation directing attention to a given fiber, it can be understood that the enzyme and the resin form a layered structure with the fiber as a base.
Description has been made in the embodiment regarding an arrangement in which the enzyme 222 is fixed first, and the resin 224 is applied afterward. However, the present invention is not restricted to such an arrangement. Also, the processing order may be swapped. That is to say, first, the resin 224 is dripped onto the holding sheet 230A, infiltrates into the interior using capillary action, and is cured. In this state, the resin 224 is distributed with a uniform density. Subsequently, a solution obtained by dissolving the enzyme 222 in phosphate buffered solution is infiltrated into the holding sheet 230A and is dried. This also provides the enzyme 222 with a uniform density.
Subsequently, as shown in
With this, as shown in
Next, description will be made regarding a specific example of the reactor 200.
With such an arrangement in which the flow paths 215 and 217 are formed, this allows the liquid sample 4 to automatically flow into the internal space 218 using capillary action without using a pump or the like. This provides the biosensor 100 with a low cost and a compact size.
As shown in
Openings are formed in the spacer 250 such that they correspond to portions of the internal space 218, the flow paths 215 and 217, the inlet 214, and the outlet 216. Furthermore, openings are formed in the upper-side film 252 such that they correspond to portions of the inlet 214 and the outlet 216. The spacer 250 is arranged such that it is interposed between the upper-side film 252 and the lower-side film 254, so as to form the flow paths 215 and 215 and the internal space 218.
An opening 256 is provided to the lower-side film 254 at a position such that it overlaps the internal space 218. Furthermore, the holding film 260 is attached so as to cover the opening 256. The reactant member 220 is attached to the holding film 260. With this, the reactant member 220 is positioned within the internal space 218.
It should be noted that, in a case in which the reactant member 220 is formed using the manufacturing method shown in
The spacer 250 is configured as a film formed of a resin material such as polyethylene terephthalate (PET) or the like. The spacer 250 has a thickness on the order of 500 μm. However, the present invention is not restricted to such an arrangement.
The upper-side film 252 is configured as a film formed of a resin material such as polyester or the like. The upper-side film 252 has a thickness on the order of 100 μm. However, the present invention is not restricted to such an arrangement. The upper-side film 252 has a lower face (that faces the spacer 250) configured as a hydrophilic face obtained by applying hydrophilic processing to its entire surface. Specific examples of the hydrophilic processing include coating the lower face of the upper-side film 252 with a surfactant or hydrophilic polymer, plasma processing, etc.
The lower-side film 254 is also configured as a film formed of a resin material such as polyester or the like as with the upper-side film 252 described above. The lower-side film 254 has a thickness on the order of 100 μm. However, the present invention is not restricted to such an arrangement. The lower-side film 254 has an upper face (that faces the spacer 250) configured as a hydrophilic face obtained by applying hydrophilic processing to its entire surface. As described above, the film 240 is formed of a resin material such as polyester or the like having a thickness on the order of 16 μm. That is to say, the film 240 has a thickness that is smaller than those of the other films 252 and 254. This allows the temperature of the reactant member 220 to be measured from the exterior with the film 240 interposed.
As shown in
The temperature sensor 110 described above is configured to be capable of detecting the difference in temperature between the reactant member 220 and the dummy reactant member 221. The temperature of the reactant member 220 corresponds to the temperature of the liquid sample 4, the reaction heat (rise in temperature) between the enzyme and the substrate, and the heat of wetting. On the other hand, the temperature of the dummy reactant member 221 corresponds to the temperature of the liquid sample 4 and the heat of wetting. Accordingly, by detecting the difference ΔT between the two temperatures, such an arrangement is capable of removing the effects of the temperature of the liquid sample 4 and the heat of wetting. This allows the component of the reaction heat (rise in temperature) to be acquired with high precision.
For example, the temperature sensor 110 may include a first thermocouple that measures the temperature of the reactant member 220 and a second thermocouple that measures the temperature of the dummy reactant member 221. The first thermocouple includes a hot junction (measurement point) that is thermally coupled to the reactant member 220. The second thermocouple includes a hot junction (measurement point) that is thermally coupled to the dummy reactant member 221. In this case, the first thermocouple and the second thermocouple may be configured to have a common cold junction (reference point). In this case, by measuring the electric potential difference between the hot junction of the first thermocouple and the hot junction of the second thermocouple, this arrangement is capable of detecting the temperature difference ΔT.
Next, description will be made regarding further features of the reactor 200.
It should be noted that the entire face that faces the reactant member 220 (upper-side wall face of the internal space) is configured to be hydrophilic. It should be noted that the entire upper face may be configured to be hydrophobic.
As described above, with the reactor 200C shown in
Furthermore, the bubble provides an effect of reducing the amount of the liquid that comes in contact with the resin. Accordingly, by controlling the bubble, this arrangement is capable of controlling the reaction amount.
It should be noted that the embodiments have been made for ease of understanding of the present disclosure. That is to say, the description thereof is by no means intended to restrict the scope of the present disclosure. Accordingly, each component disclosed in the embodiments described above is intended to include all changes of design and all equivalents that belong to the technical scope of the present invention.
For example, multiple kinds of enzymes may be immobilized on the same holding sheet. As a specific example in this case, in a case in which glucose is employed as the substrate and glucose oxidase is employed as the enzyme, catalase may be held on the holding sheet in addition to the glucose oxidase. Hydrogen peroxide is generated due to the contact reaction between glucose and glucose oxidase. By further providing a contact reaction between this hydrogen peroxide and catalase, such an arrangement is capable of providing increased reaction heat ΔT.
Also, an arrangement may be made in which the liquid sample contains an enzyme, and a substrate that corresponds to the enzyme is immobilized on the holding sheet. Specific examples of this case include an arrangement in which acid phosphatase is employed as the enzyme, and 1-naphthyl phosphate is employed as the substrate.
Also, a liquid that differs from body fluids may be employed as the liquid sample. Examples of such liquids include liquids obtained from vegetables, fruit, or seaweed.
Description has been made above in the embodiments regarding the biosensor 100 including the temperature sensor 110 and the reactor 200 configured as separable components. However, the structure of the biosensor is not restricted to such an arrangement. The technique according to the present disclosure may be applied to a biosensor including a flow path member and a sensor member integrated as a single unit.
While the preferred embodiments of the present disclosure have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-176740 | Oct 2020 | JP | national |
This application is a continuation under 35 U.S.C. § 120 of PCT/JP2021/035911, filed Sep. 29, 2021, which is incorporated herein by reference, and which claimed priority to Japanese Application No. 2020-176740, filed Oct. 21, 2020, the entire content of which is also incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2021/035911 | Sep 2021 | US |
| Child | 18304858 | US |
